Most important biological processes are mediated by "protein machines," very large multiprotein complexes whose component polypeptides often affect one anther's properties through direct interactions. Traditional biochemical methods are ill suited to study all aspects of the largest protein machines, such as RNA polymerase holoenzymes, splicosomes, and nuclear pore complexes, because classical reconstitution of the complex from purified proteins is unfeasible. Therefore, new methods are necessary to derive detailed, information about the mechanism of faction of these machines in their intact form. This proposal addresses the problem of elucidating the organization of proteins in large complexes and understanding how the array of protein-protein interactions might change during the course of the reaction in question. A novel approach noncovalent affinity modification will be developed that involves specific delivery of a small molecule with a latent reactive group to a particular protein in a large complex via noncovalent interactions. Specific delivery of the reactive agent will be achieved by fusing to the protein of interest at the genetic level a peptide (selected from a library) that binds the small molecule with high affinity and specificity. When activated, the small molecule will react with proximal proteins, resulting in their covalent modification. If a sensitive method exists to detect these products, subsequent analysis of the mixture under denaturing conditions will reveal which proteins were nearby the small molecule-docking site in the complex. Iterations of this type of experiment would allow the organization of large protein machines to be mapped out and experiments conducted at different times in the reaction cycle could reveal how the organization of the complex might change with time. While this work is directed towards the development of a new technology and is not focused on any particular disease state per se, the health-related impact of the development of this methodology would be significant. Multiprotein complexes mediate essentially all processes that are thought to lead to cancer when they malfunction, including the decoding of genetic information and regulation of the cell cycle.